SEARCH

SEARCH BY CITATION

Non-technical summary

  1. Top of page
  2. Non-technical summaryAbstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Activation of the sympathetic nervous system hastens the rate of progression and severity of chronic heart failure (CHF). Chemically sensitive nerves in the carotid body (CB) that stimulate sympathetic nerve activity become overly active in CHF and contribute to this phenomenon. The stimulus for activation of these CB chemoreceptors is not known. Blood supply to tissues is impaired due to the failing heart. In this study we tested whether a chronic reduction in blood flow to the CB may contribute to altered CB chemoreceptor function. The results show that changes that occur in CB chemoreceptor function during CHF are identical to those that occur if blood flow is simply reduced to the CB for several weeks. The results suggest that chronic impairment of blood flow may be the key step in the pathophysiological events that cause sympathetic nervous system activation in heart failure.

Abstract

Peripheral chemoreflex sensitivity is potentiated in clinical and experimental chronic heart failure (CHF). Blood supply to tissues is inevitably reduced in CHF. However, it remains poorly understood whether the reduced blood flow is the cause of increased peripheral chemoreflex sensitivity in CHF. This work highlights the effect of chronically reduced blood flow to the carotid body (CB) on peripheral chemoreflex function in rabbits. In pacing-induced CHF rabbits, blood flow in the carotid artery was reduced by 36.4 ± 5.2% after 3 weeks of pacing. For comparison, a similar level of blood flow reduction was induced by carotid artery occlusion (CAO) over a similar 3 week time course without pacing. CB blood supply was reduced by similar levels in both CHF and CAO rabbits as measured with fluorescent microspheres. Compared with sham rabbits, CAO enhanced peripheral chemoreflex sensitivity in vivo, increased CB chemoreceptor activity in an isolated CB preparation and decreased outward potassium current (Ik) in CB glomus cells to levels similar to those that were observed in CHF rabbits. In CAO CB compared to sham, neural nitric oxide (NO) synthase (nNOS) expression and NO levels were suppressed, and angiotensin II (Ang II) type 1 receptor (AT1-R) protein expression and Ang II concentration were elevated; these changes were similar to those seen in the CB from CHF rabbits. A NO donor and AT1-R antagonist reversed CAO-enhanced chemoreflex sensitivity. These results suggest that a reduction of blood flow to the CB is involved in the augmentation of peripheral chemoreflex sensitivity in CHF.

Abbreviations 
Ang II

angiotensin II

AP

action potential

AT1-R

angiotensin II type 1 receptor

CB

carotid body

CAO

carotid artery occlusion

CABF

carotid artery blood flow

DF

discharge frequency

FI

Fluorescence intensity

FM

fluorescent microsphere

Ik

outward potassium current

MBP

mean blood pressure

nNOS

neural nitric oxide synthase

NO

nitric oxide

RSNA

renal sympathetic nerve activity

SNAP

S-nitroso-N-acetyl-l-penisillamine

TH

tyrosine hydroxylase

inline image

minute ventilation

Introduction

  1. Top of page
  2. Non-technical summaryAbstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Activation of the sympathetic nervous system is a major contributor to the progression of morbidity and ultimate mortality of chronic heart failure (CHF) (Leimbach et al. 1986; Francis et al. 1990; Floras, 1993; Mark, 1995; Esler et al. 1997). It is now known that multiple reflexes and central adjustments contribute to the altered sympathetic nervous system in CHF. In particular, enhanced chemoreflex-mediated activation of sympathetic outflow occurs in patients and experimental animals with CHF (Chugh et al. 1996, 1997; Sun et al. 1999a,b; Schultz & Li, 2007).

Our studies have documented that an augmented afferent input from the carotid body (CB) chemoreceptors mediates the enhancement of peripheral chemoreflex function in pacing-induced CHF rabbits (Sun et al. 1999a), and this enhanced input contributes to increased sympathetic activation in the CHF state (Sun et al. 1999b). The specific mechanisms responsible for the enhanced chemosensory input from the CB in CHF are beginning to come to light. Our studies indicate activation of the local angiotensin II (Ang II) system (Li & Schultz, 2006, 2007; Li et al. 2007) and a decreased neural nitric oxide synthase–nitric oxide (nNOS–NO) pathway (Li et al. 2004, 2005, 2010; Ding et al. 2008) in the CB are involved in the augmentation of CB chemoreceptor activity induced by CHF.

CHF reduces blood supply to tissues due to depressed cardiac function (Rajagopalan et al. 1984; Wanless et al. 1987; Spinale et al. 1992; Seymour et al. 1994; Krombach et al. 1998; Choi et al. 2006). Myocardial, renal and skeletal muscle blood flows are reduced in heart failure induced by rapid left ventricular pacing in dogs (Spinale et al. 1992; Seymour et al. 1994) and pigs (Krombach et al. 1998). In clinical studies, whole or regional cerebral blood flow is significantly reduced in CHF patients compared to that in normal subjects (Rajagopalan et al. 1984; Choi et al. 2006). Chronic low cardiac output is associated with a 25% reduction in cerebral blood flow in cardio-myopathic rabbits (Wanless et al. 1987). These studies strongly support the concept that blood supply to the CB is reduced during CHF. Although studies have shown that an acute reduction in blood flow in the carotid artery has little or no effect on chemoreceptor activity (Biscoe et al. 1970; Lahiri, 1980), it is not clear whether a chronic blood flow reduction affects CB chemoreceptor and reflex function.

The goals of the present study were to determine whether chronically reduced carotid artery blood flow (CABF) induces an enhanced CB chemoreflex sensitivity and chemoreceptor activity, and subsequently, whether changes in the CB Ang II system and nNOS–NO pathway are responsible for the altered CB chemoreceptor function induced by reduced CABF.

Methods

  1. Top of page
  2. Non-technical summaryAbstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

General surgical preparation and induction of chronic heart failure (CHF)

Sixty male New Zealand White rabbits weighing 2.5–3.5 kg were randomly assigned to sham, carotid artery occlusion (CAO) and CHF groups. Rabbits were housed in individual cages under controlled temperature and humidity and a 12:12 h dark–light cycle and fed standard rabbit chow with water available ad libitum. The experimental protocols were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health (NIH Publication No. 85-23, revised 1996) and the American Physiological Society's Guide for the Care and Use of Laboratory Animals.

CHF was induced by pacing and monitored by weekly echocardiograms as previously described (Sun et al. 1999b; Ding et al. 2010). Rabbits were anaesthetized with a cocktail of 5.8 mg kg−1 xylazine, 35 mg kg−1 ketamine and 0.01 mg kg−1 atropine given i.m., intubated and connected to a small animal anaesthesia respiration unit using 2.0–5.0% inhalation isofluorane with oxygen for the duration of the surgery. Using sterile technique, a pressure telemetry unit was implanted into a branch of the femoral artery and advanced into the iliac artery or into the abdominal aorta. A left thoracotomy was performed to implant a pin electrode on the basis of left ventricle for pacing. The leads of pacing electrodes were tunnelled subcutaneously and fixed on the back. Rabbits were given an antibiotic regimen consisting of 5 mg kg−1 Baytril i.m. repeated daily for 5 days. All experiments were conducted after the animals had fully recovered for 7–10 days from surgery.

The rapid ventricular pacing model of inducing CHF was carried out using a pacemaker of our design. We initially began pacing at a rate of 340 bpm, which was held for 7 days, and then the rate was gradually increased to 380 bpm, with an increment of 20 bpm each week. Each rabbit was checked daily to insure chronic pacing. Rabbits were paced continually for 3–4 weeks. The progression of CHF was monitored by weekly echocardiograms (Siemens/Acuson Sequoia 512C with a 4 MHz probe, Siemens Medical Solutions USA, Inc., Malvern, PA, USA), with the pacemaker turned off for at least 30 min before the recordings were started. CHF was characterized by >40% reduction in ejection fraction and fraction of shortening.

Experimental groups

Studies were divided into three groups: (1) sham group: sham operations without pacing or inflation of carotid occluders; (2) CHF group: pacing without inflation of carotid occluders; (3) CAO group: inflation of carotid occluders without pacing.

Reduction of carotid artery blood flow (CABF) induced by carotid occluders

Two weeks prior to pacing or CAO, Silastic occluders and ultrasonic flow probes (Transonic Systems Inc., Ithaca, NY, USA) were implanted bilaterally on the carotid arteries to chronically reduce (CAO group) and monitor CABF, respectively. To achieve the desired reduction in CABF in the CAO group, saline was injected into the occluders until the desired flow was achieved to mimic the reduction of mean CABF observed in CHF rabbits, then checked and adjusted daily if needed to maintain the expected level. The occluders were not inflated in the CHF group.

To measure CABF, the flow probes were connected to a flow meter (Transonic system T106) and calibrated. The pulse and mean CABF were measured in ml min−1 and fed into an analog-to-digital converter (PowerLab; ADInstruments, Colorado Springs, CO, USA) attached to a computer. Changes in CABF were expressed as percentage of baseline. Rabbits were acclimated to the lab surroundings prior to the studies.

Measurement of CB flow by fluorescence microspheres

At the end of the study of some rabbits, the CB blood flow was determined by the fluorescent microspheres (FMs) method (Thein et al. 2002). Briefly, the left ventricle was catheterized, and correct positioning of the catheter tip was ascertained by observing the typical waveform of the left ventricular pressure curve. FMs (Molecular Probes/Invitrogen, Carlsbad, CA, USA) were injected at a dosage of 1 × 106 spheres (kg body weight)−1 in 3 ml during 30 s. Arterial reference blood samples were withdrawn at 0.5 ml min−1 from the inferior aorta via the right femoral artery. After 10 min, CB tissue was taken and weighed. Fluorescence intensity (FI) of reference blood sample (FIreference) and CB sample (FIsample) was measured by fluorescence spectroscopy to get the ratio of FIsample/FIreference. Blood flow was calculated by multiplying blood sample withdrawal rate and CB weight and expressed as ml min−1 (100 g tissue)−1.

Chemoreflex evaluation with renal sympathetic nerve activity (RSNA) and minute ventilation (inline image)

RSNA and inline image were recorded in conscious rabbits as described in our previous studies (Sun et al. 1999b; Li et al. 2005; Ding et al. 2010). The left kidney was exposed retroperitoneally, and a branch of renal nerve was separated from the renal plexus and the surrounding connective tissues. A pair of stainless steel stranded Teflon coated recording electrodes was placed around the nerve branch. The nerve–electrode junction was insulated electrically from the surrounding tissues and covered with a fast setting silicone (Kwik-Sil, World Precision Instruments, Sarasota, FL, USA). A ground wire was sutured to a nearby muscle before the incision was closed. The electrode wires were tunnelled beneath the skin to exit on the upper back. Rabbits were allowed to recover for 3 days, depending on the quality of RSNA, before experiments commenced. RSNA was recorded using a Grass P511 differential amplifier (Grass Technologies/Astro-Med Inc., West Warwick, RI, USA) and a storage oscilloscope. The RSNA was filtered at a bandwidth of 100 Hz to 3 kHz. The nerve signal was also fed to an audio amplifier and loudspeaker. The nerve signal was rectified and integrated, and both raw and integrated signals were recorded. All analog signals were captured and digitized by using a PowerLab (Model 8S) data acquisition system. RSNA was expressed as the percentage maximum, and maximal RSNA was determined in each rabbit by an intravenous bolus injection of sodium nitroprusside (100 μg kg−1) at the end of the experiment.

Tidal volume (TV) and breathing rate (BR) were determined by unrestrained plethysmography. Minute ventilation (inline image) was defined as TV × BR and was normalized to body weight. Rabbits were placed in a Plexiglas chamber (volume 11 litres) with exit ports for catheters and renal nerve electrodes. The chamber was sealed, except for an inlet and outlet port that allowed a continuous flow of air through the chamber. Different gas mixtures could be easily passed through the chamber to alter blood gases. TV was measured by temporarily (30 s) sealing the air ports and measuring the pressure changes in the sealed chamber by using an MP45 differential pressure transducer and amplifier (Validyne Engineering Corp., Northridge, CA, USA) connected to the PowerLab analysis system. The chamber was sealed only for short intervals (30 s) to prevent appreciable changes in the composition of air in it. All ventilatory gases were saturated with water vapour by passage through a warmed bubbling chamber.

Changes in RSNA and inline image in response to stimulation of peripheral chemoreceptors were measured in sham, CHF and CAO rabbits in the conscious resting state. Peripheral chemoreceptors were stimulated preferentially by allowing the rabbits to breathe graded mixtures of hypoxic gas under isocapnic conditions. Different concentrations of O2 with balance of N2 were delivered into the chamber in the following sequence: 21% O2 (normoxia), 15% O2 (mild hypoxia), and 10% O2 (severe hypoxia). Each stimulation was held until a steady response was achieved for 3 min. An arterial blood sample was taken from the arterial catheter for the measurement of inline image, inline image and pH. Because hypoxic stimulation of ventilation induces hyperventilatory hypocapnia, 2–4% CO2 was added to the hypoxic gases to maintain relatively constant inline image during hyperventilation. Sufficient recovery time at 21% O2 was allowed between stimuli to ensure that all variables returned to baseline levels. In five CAO rabbits, inline image responses to isocapnic hypoxia were determined sequentially at pre-CAO, CAO 1 day, CAO 1 week, 2 weeks and 3 weeks.

Recording of afferent discharge of CB chemoreceptors

Single unit action potentials were recorded from CB chemoreceptor fibres in the carotid sinus nerve as in previous studies (Sun et al. 1999a; Li et al. 2005; Ding et al. 2010). In some of the CAO animals, one carotid artery was chronically occluded to reduce CABF while the contralateral CB remained unobstructed to serve as a paired control. Briefly, the left and/or right carotid sinus region was vascularly isolated sequentially and perfused with Krebs–Henseleit solution (in mm: 120 NaCl, 4.8 KCl, 2.0 CaCl2, 2.5 MgSO4, 1.2 KH2PO4, 25 NaHCO3 and 5.5 glucose; 10 ml min−1, 37°C). Perfusate was bubbled with an O2–CO2–N2 gas mixture to maintain inline image at 100–110 mmHg, inline image at 30–35 mmHg, and pH 7.4 as the normoxic condition. Flow through the isolated sinus was set at 10 ml min−1 at a perfusion pressure of 80 mmHg and inline image of the perfusate was altered by bubbling with gas mixtures (containing the different O2 concentrations, a constant fraction of CO2 and a balance of N2) to achieve a inline image of 55–65 and 35–45 mmHg, respectively. All chemicals were obtained from Sigma-Aldrich Chemical Co., St Louis, MO, USA.

The carotid sinus nerve was exposed and transected near the petrosal ganglion to interrupt neural efferents to the CB. The nerve was covered with mineral oil, and fine slips of nerve filaments were placed on a silver electrode. Impulses were amplified with a bandwidth of 100 Hz to 3 kHz (Grass P511), displayed on an oscilloscope (2120 oscilloscope, B&K Precision Corp., Yorba Linda, CA, USA), and fed into a rate meter (FHC Inc., Bowdoin, ME, USA) whose window discriminators were set to accept potentials of the particular amplitude. Bundles that had one, or at most two, easily distinguishable active fibres were used. Chemoreceptor afferents were identified by their sparse and irregular discharge at normoxia and by their response to hypoxia and NaCN.

Isolation and recording of outward K+ currents (IK) in CB glomus cells

The procedures for isolation, identification of glomus cells, and recording of its K+ currents (IK) were performed as described previously (Li et al. 2004; Ding et al. 2010). Briefly, the carotid bifurcations on both sides were removed surgically from rabbit anaesthetized with the cocktail described above. The rabbits were then killed with an intravenous injection of 150 mg kg−1 sodium pentobarbital. The removed CB tissue was placed in ice-cold Ca2+/Mg2+-free solution (mm: 140 NaCl, 5 KCl, 10 Hepes, 5 glucose, pH 7.2), and then minced with microscissors and subjected to a two-step enzymatic digestion protocol. Each step lasted for 30 min at 37°C. The enzymatic solution for the first step of digestion contained trypsin (type II, 2 mg ml−1) and collagenase (type IV, 2 mg ml−1). That for the second step contained collagenase (4 mg ml−1) and bovine serum albumin (5 mg ml−1). Other solutes in the solutions used for enzymatic digestion were the same as the above Ca2+/Mg2+-free solution. The digested tissue fragments were gently triturated for 1 min every 10 min during the process of digestion. CB cells were obtained after centrifugation of the digested tissue at 150 g for 5 min. The isolated cells were then resuspended in culture medium and plated onto culture wells. The culture medium consisted of a 50/50 mixture of Delbecco's modified Eagle's medium (DMEM) and Ham's F12 medium supplemented with antibiotics and 10% fetal bovine serum. The CB cells including glomus cells, other neural cells and vascular cells were cocultured at 37°C in a humidified atmosphere of 95% air–5% CO2 and studied within 24 h of dissociation. In some of the CAO animals for this experiment, one carotid artery was chronically occluded to reduce CABF while the contralateral CB remained unobstructed to serve as a paired control.

Glomus cells were identified by their characteristic spherical shape. Using the patch clamp technique described below, patched cells were initially superfused with an extracellular solution of the following composition (mm): 140 NaCl, 5.4 KCl, 2.5 CaCl2, 0.5 MgCl2, 5.5 Hepes, 11 glucose, 10 sucrose, pH 7.4 to identify glomus type I cells, which exhibit a NaV current. Once a type I cell was confirmed, the extracellular solution was changed to solution containing 0.5 μm TTX (a Na+ channel blocker) to record IK. The pipette solution contained the following composition (mm): 105 potassium aspartate, 20 KCl, 1 CaCl2, 10 EGTA, 5 Mg-ATP, 10 Hepes and 25 glucose, pH 7.2. All chemicals were obtained from Sigma-Aldrich.

All experiments were performed at 22°C. Patch pipettes had resistances of 4–6 MΩ when filled with intracellular solution. Currents were measured in the whole-cell configuration of the patch-clamp technique using a Warner PC-501A patch-clamp amplifier (Warner Instrument Corp. Hamden, CT, USA) and the pCLAMP 8.1 program (Molecular Devices, Sunnyvale, CA, USA). Current traces were sampled at 10 kHz and filtered at 5 kHz. Holding potential was −80 mV. Current–voltage (I–V) relations were elicited by 400 ms test pulses from −80 mV to +80 mV applied in 10 mV increments (5 s between steps). Peak currents were measured for each test potential and plotted against the corresponding test potential.

Measurement of Ang II and NO concentration in the CB

Tissue homogenate was prepared from CB samples. CB Ang II concentrations were measured with an Ang II 125I radioimmunoassay kit (Bühlmann Laboratories, Schönenbuch, Switzerland). The final Ang II concentration was counted with a 1470 Automatic Gamma Counter (Perkin Elmer, Shelton, CT, USA) and calculated with a standard curve generated for each experiment.

NO concentration was determined by the Apollo 1000 NO sensor (World Precision Instruments). The NO probe (ISO-NOPF100) was calibrated based on a solution of 0.1 m CuCl2 using the NO donor S-nitroso-N-acetyl-l-penicillamine (SNAP) at concentrations of 5, 25, 50, 100, 200 and 400 nm. The NO concentration in the CB was calculated with this SNAP standard curve and normalized by total protein (BCA protein assay kit, Pierce/Thermo Fisher Scientific, Rockford, IL, USA).

Immunofluorescence detection and Western blot analysis in the CB

We performed immunofluorescence detection and Western blot analysis as described in our previous studies (Ding et al. 2008, 2009, 2010). For immunofluorescence staining, CB sections, 10 μm thick, were mounted on pre-coated glass slides for AT1-R, nNOS and tyrosine hydroxylase (TH) detection. TH served as a marker of glomus type I cells. CB sections were blocked with 10% normal donkey serum for 1 h and incubated with primary anti-AT1-R (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-nNOS (Abcam, Cambridge, MA, USA) and anti-TH antibodies (Sigma-Aldrich) overnight at 4°C, followed by incubation with appropriate secondary antibody (Molecular Probes/Invitrogen) for 1 h at room temperature. Slides were observed under a Leica fluorescence microscope with appropriate excitation/emission filters and pictures were captured by a digital camera system. No staining was observed with the procedure described above using phosphate-buffered saline instead of the primary antibody. For protein measurement by Western analysis, CB tissue was rapidly removed and immediately frozen in dry ice and stored at −80°C until analysed. The concentration of total protein extracted from CB tissue was measured using a BCA protein assay kit (Pierce/Thermo Fisher Scientific). After electrophoresis, proteins were electrophoretically transferred onto the polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was probed with primary antibody (1:1000 dilutions of anti-AT1-R or anti-nNOS) and secondary antibody (1:5000 dilutions) IgG-horseradish peroxidase (Pierce), respectively, and then treated with enhanced chemiluminescence substrate (Pierce). The bands in the membrane were visualized and analysed using UVP BioImaging Systems (UVP, LLC, Upland, CA, USA). The protein intensity was normalized to β-tubulin intensity, which did not vary among groups.

Contribution of Ang II and NO to CB chemoreflex function after CAO

Protocol 1. For evaluation of CB chemoreflex activation of RSNA in conscious rabbits, the RSNA–inline image curve was recorded before and after bolus administration of the AT1-R antagonist losartan (0.5 mg kg−1i.v.) or infusion of the NO donor SNAP (1.2 μg kg−1 min−1i.v.). To minimize the hypotensive effect of SNAP on the baroreflex, phenylephrine (0.2–1.0 μg kg−1 min−1i.v.) was infused simultaneously with SNAP to keep arterial pressure at the control level.

Protocol 2. For evaluation of CB chemoreceptor sensitivity in the isolated CB preparation, the inline image–chemoreceptor afferent discharge frequency (DF) relationship was recorded before and after perfusion of the CB with losartan (1.0 μm) or SNAP (100 μm).

Data analysis

All values are presented as means ±s.e.m. Statistical significance was determined by a two-way ANOVA, followed with a Bonferroni procedure for post hoc analysis for multiple comparisons. Statistical significance was accepted when P < 0.05. A power analysis was conducted to assess whether the sample size was sufficient.

Results

  1. Top of page
  2. Non-technical summaryAbstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Cardiac function in sham, CHF and CAO rabbits

Impairment of left ventricular function after 3 weeks of cardiac pacing in the CHF group (340 bpm in 1st week, 360 bpm in 2nd week, 380 bpm in 3rd week) is shown in Table 1. The rate of induction of CHF and degree of LV dysfunction were consistent with our previous studies (Sun et al. 1999b; Li et al. 2004, 2005, 2006, 2007, 2008; Li & Schultz, 2006; Ding et al. 2008, 2009, 2010). CABF reduction by CAO for 3 weeks did not significantly affect cardiac function compared with that from sham rabbits.

Table 1.  Body weight, ventricular weight and cardiac function in sham, chronic heart failure (CHF) and carotid artery occlusion (CAO) rabbits
 ShamCHFCAO
  1. Echocardiography was performed with the pacer off for at least 30 min. Data are means ±s.e.m.; BW, body weight; LVW, left ventricular weight; LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter; FS, fractional shortening; EF, ejection fraction. *P < 0.05 vs. sham.

n202020
BW (kg)3.7 ± 0.33.8 ± 0.23.6 ± 0.2
LVW/BW (g kg−1)1.4 ± 0.11.9 ± 0.1*1.4 ± 0.2
LVEDD (mm)13.6 ± 0.417.6 ± 0.5*13.2 ± 0.3
LVESD (mm)9.2 ± 0.512.3 ± 0.5*9.0 ± 0.4
FS (%)39.3 ± 0.420.2 ± 1.6*38.8 ± 1.0
EF (%)75.3 ± 1.242.2 ± 1.0*74.5 ± 0.8

Decreased carotid blood flow induced by CHF and CAO

Cardiac pacing evoked a marked decrease in CABF and mean blood pressure (MBP) (Fig. 1A–C). The salient features of this change in CABF were an immediate and sustained decrease in CABF of about 20–36% of baseline followed by incomplete autoregulation of CABF (∼80% of control) during the first 3 days of pacing. Then over the course of 1–3 weeks of pacing, a progressive decline in CABF occurred as cardiac function deteriorated. CABF was reduced by 36 ± 5.2% at 3 weeks pacing (Fig. 1D). CABF recovered to normal levels when the pacer was turned off for 30 min at earlier pacing periods (Fig. 1D); however, the decreased CABF was sustained without pacing at the later periods as a manifestation of induction of CHF.

image

Figure 1. Pacing decreases carotid artery blood flow (CABF) A, an original recording of pulsatile CABF (PCABF), mean CABF (MCABF) and arterial blood pressure (ABP). B and C, summarized changes of MCABF and mean blood pressure (MBP) over the pacing duration (n= 7). D, MCABF changes expressed as a percentage of baseline with pacer on and pacer off (30 min). E, MCABF changes expressed as a percentage of baseline induced by CHF (n= 7) or by carotid artery occlusion (CAO, n= 15). B–D, means ±s.e.m. *P < 0.05 compared with baseline.

Download figure to PowerPoint

In CAO rabbits, we mimicked the same degree of CABF reduction seen with pacing over the course of 3 weeks using adjustable cuff occluders on the carotid arteries (Fig. 1E). Chronic CAO did not alter resting MBP (79.5 ± 3.0 mmHg pre-CAO vs. 80.2 ± 2.4 at 3 weeks CAO).

Blood flow in the CB from CHF rabbits at 3 weeks pacing was 40% of that in sham rabbits (Fig. 2, P < 0.05). CB blood flow in the CAO group at 3 weeks was similar to that of CHF rabbits (Fig. 2). The CB flow in sham rabbits was 1203 ± 126 ml min−1 (100 g)−1 (17 ± 2.6 μl min−1), which is similar to that reported in cats (Barnett et al. 1988) and rabbits (McCloskey & Torrance, 1971).

image

Figure 2. CB blood flow measured by fluorescence microspheres from sham, CHF and CAO rabbits Flow measurements (n= 5) and data in subsequent figures (unless noted otherwise) were taken at the end of a 3 week period of CAO, pacing (CHF) or sham intervention. Data are means ±s.e.m., *P < 0.05 compared with sham.

Download figure to PowerPoint

CAO enhanced CB Chemoreflex sensitivity in vivo

CAO induced a gradual functional enhancement of hypoxic ventilatory CB chemoreflex sensitivity over the course of 3 weeks of reduced CABF flow, which reached statistical significance (P < 0.05) by the second week of CAO (Fig. 3). As illustrated in Fig. 3, 1 day of CAO did not affect the inline imageinline image curve. Similarly, acute reductions of CABF by 30–40% over the first few hours did not alter CB hypoxic ventilatory reflex sensitivity (not shown). Thus the enhancement of chemoreflex sensitivity was not related to an acute reduction in CABF per se.

image

Figure 3. Effects of normoxic and hypoxic states on minute ventilation before and during CAO n= 5. inline image, fraction of oxygen in inspired air. Data are means ±s.e.m. *P < 0.05 CAO 2 weeks, and 3 weeks compared with pre-CAO.

Download figure to PowerPoint

CAO for 3 weeks increased baseline RSNA (normoxia) and enhanced RSNA response to hypoxia compared with that in sham rabbits (Fig. 4A and B), which were similar in magnitude to those observed in CHF animals.

image

Figure 4. Effects of normoxic and hypoxic states on renal sympathetic nerve activity (RSNA) and CB chemoreceptor discharge in sham, CHF and CAO rabbits A, representative recording of RSNA; B, RSNA response to hypoxia (n= 12); C, representative recording of CB chemoreceptor afferent discharge; D, CB chemoafferent response to hypoxia (n= 6). DF, discharge frequency; AP, action potential; inline image, arterial oxygen partial pressure. Data are means ±s.e.m. *P < 0.05 CHF and CAO compared with sham group.

Download figure to PowerPoint

CAO increased CB chemoreceptor sensitivity

To assess whether the enhanced chemoreflex function during CAO is associated with altered CB chemoreceptor activity, discharge from CB chemoreceptor fibres was recorded in the isolated CB. CAO for 3 weeks increased CB chemoreceptor activity at normoxia and increased the response to hypoxia (Fig. 4C and D) compared with sham rabbits. These effects were similar in magnitude to those in CHF animals.

CAO decreased Ik in CB glomus cells

IK was similarly decreased in the CB glomus cells from CAO and CHF rabbits compared to sham (Fig. 5), which is consistent with the known effect of CHF to decrease Ik activity in CB glomus cells (Li et al. 2004; Li & Schultz, 2006, 2007; Ding et al. 2009). The glomus cell size evaluated by member capacitance was not different among sham (2.81 ± 0.23 pF), CHF (3.10 ± 0.21 pF) and CAO (2.92 ± 0.30 pF) groups.

image

Figure 5. Outward K+ current (IK) in CB glomus cells from sham, CHF and CAO rabbits A, representative IK recording evoked by 400 ms depolarizing test pulses from a holding potential of −80 to +80 mV in 10 mV increments; B, peak current–voltage relationship curves. Data are means ±s.e.m. n= 8 cells from 4–6 carotid bodies each group. *P < 0.05 CHF and CAO compared with sham group.

Download figure to PowerPoint

CAO downregulated CB nNOS-NO

NOS protein expression and NO production are markedly decreased in the CB from CHF rabbits (Li et al. 2005, 2008; Ding et al. 2008, 2009, 2010). In the CB from CAO rabbits, nNOS localized to glomus cells (Fig. 6A), nNOS protein expression (Fig. 6B and C) and NO production (Fig. 6D) were significantly decreased compared with that in the CB from sham rabbits. There were no differences in nNOS expression and NO production in the CB from CAO rabbits compared with CHF CB.

image

Figure 6. Expression of CB nNOS and nitric oxide level in sham, CHF and CAO rabbits A, co-localization of tyrosine hydroxylase (TH) and nNOS in the CB. Green immunofluorescence image for TH, red immunofluorescence image for nNOS, and merged image, yellow, for overlap of TH and nNOS. B, representative bands from Western blot analysis. C, total relative nNOS protein expression (n= 4). D, nitric oxide level in the CB (n= 4). Data are means ±s.e.m. *P < 0.05 compared with sham group.

Download figure to PowerPoint

CAO upregulated CB Ang II–AT1-R

AT1-R protein expression and Ang II production are markedly increased in the CB from CHF rabbits (Li et al. 2006, 2008; Ding et al. 2009, 2010). CAO increased CB AT1-R expression localization within glomus cells compared with that in sham CB, and there was no difference between CHF and CAO groups (Fig. 7AC). Similar to AT1-R protein, CAO elevated Ang II concentration in the CB compared with that in sham CB, and the enhanced Ang II concentration was not different from that in CHF CB (Fig. 7D).

image

Figure 7. Expression of CB AT1-R and Ang II concentration in sham, CHF and CAO rabbits A, co-localization of TH and Ang II type 1 receptor (AT1-R) in the CB. Green immunofluorescence image for TH, red immunofluorescence image for AT1-R, and merged image, yellow, for overlap of TH and AT1-R. B, representative bands from Western blot analysis. C, total relative AT1-R protein expression (n= 4). D, Ang II concentration in the CB (n= 4). Data are means ±s.e.m. *P < 0.05 compared with sham group.

Download figure to PowerPoint

SNAP and losartan reversed CB reflex and afferent function in CAO rabbits

The NO donor SNAP (while maintaining a constant arterial blood pressure by phenylephrine infusion) markedly decreased the CAO-enhanced chemoreflex in vivo (Fig. 8A) and CAO-induced CB chemoreceptor hyperactivity in isolated CB preparations (Fig. 8C). Similarly, the AT1-R antagonist losartan significantly attenuated CAO-enhanced chemoreflex in vivo (Fig. 8B) and CB chemoreceptor activity in the isolated CB preparation (Fig. 8D).

image

Figure 8. Effects of NO donor and AT1-R blocker on CB chemoreflex and chemoreceptor responses to hypoxia in CAO rabbits A and B, RSNA responses to hypoxia in vivo before and after i.v. administration of NO donor S-nitroso-N-acetyl-l-penicillamine (SNAP,1.2 μg kg−1 min−1i.v., n= 6) (A) and AT1-R blocker losartan (0.5 mg kg−1i.v., n= 5) (B). C and D, CB chemoreceptor discharge responses to hypoxia in the isolated CB before and during perfusion with SNAP (100 μm, n= 5) (C) and losartan (1 μm, n= 4) (D). Data are means ±s.e.m. *P < 0.05 compared with control.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Non-technical summaryAbstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Our previous studies have established that CHF enhances CB chemoreceptor activity and chemoreflex activation and have explored important mechanisms of action including the upregulation of Ang II signalling (Li & Schultz, 2006, 2007; Schultz & Li, 2007) and downregulation of the NOS system in the CB over the course of the development of CHF (Li et al. 2004, 2005, 2010; Ding et al. 2008). However, the primary stimulus responsible to initiating these changes in the CB over the course of the development of CHF has not yet been addressed. The present study sheds light on the role of a chronic reduction of CB blood flow to these effects.

Reduction of CB blood flow induced by CHF and CAO

CHF inevitably reduces blood supply to tissues including myocardial, renal, skeletal muscle and cerebral blood flows (Rajagopalan et al. 1984; Wanless et al. 1987; Spinale et al. 1992; Seymour et al. 1994; Choi et al. 2006). Our present study shows that CABF and CB blood flow are similarly reduced in CHF induced by rapid left ventricular pacing in conscious rabbits. Our model of chronically reducing CABF by carotid artery occlusion mimicked the same degree of CB blood flow reduction (40%) as observed in CHF rabbits.

Enhanced sympathetic excitation by CB chemoreflex in CAO and CHF states

Sympathetic activation is accepted as an important factor in the progression of CHF (Mark, 1995; Zucker et al. 1995; Esler et al. 1997). A unified hypothesis for the sympathetic activation implicates alterations in multiple afferent and central neural pathways that control sympathetic activity. An augmented CB chemoreflex sensitivity is one important reflex pathway that contributes to the elevated sympathetic activation in patients (Chugh et al. 1996; Chua et al. 1997) and experimental animals (Sun et al. 1999b; Li et al. 2005, 2006) with CHF. In our CAO model, we observed an enhanced CB chemoreflex and sympathetic hypersensitivity similar to that induced by CHF (Fig. 4A and B). These results suggest that chronic CABF reduction indeed contributes to CB chemoreflex hypersensitivity and the enhanced sympathetic excitation in CHF.

Increased chemoreceptor activity in CAO and CHF states

Our previous work has established that the discharges of CB chemoreceptor afferents in the carotid sinus nerve at normoxia and in response to isocapnic hypoxia are enhanced in CHF rabbits (Sun et al. 1999a; Li et al. 2005, 2006). After 3 weeks CAO, the activity of CB chemoreceptors in the normoxic state and the magnitude of the response to hypoxia were enhanced similarly to that seen in CHF rabbits. These results suggest that the enhanced CB chemoreflex function induced by CAO is mediated by elevated activity of CB chemoreceptors, similar that documented for CHF (Sun et al. 1999b; Li et al. 2005, 2006, 2007, 2008; Ding et al. 2008, 2009, 2010). However, we cannot exclude a potential complication of the reflex study, in which a reduction in CABF may affect central pathways of the chemoreflex due to reduced cerebral blood flow. In addition, the CAO may have unloaded carotid sinus baroreceptors to influence central integration of the chemoreflex. However, baroreflex activation was not likely to be a major factor as resting blood pressure was not affected by the procedure. Nevertheless, the present study clearly demonstrates that a chronic reduction in CB blood flow induces an enhanced activity from CB chemoreceptors, which is independent of these central nervous variables. This effect is likely to be the contributing factor to the similarly enhanced CB chemoreceptor activity observed in CHF.

Depressed IK of glomus cell in CAO and CHF states

Glomus cells, which lie in synaptic apposition with afferent nerve ending in the CB, are the initial site of sensory transduction in the CB (Eyzaguirre & Fidone, 1980). Oxygen sensitive K+ channel activity plays an important role in glomus cell sensory transduction (López-Barneo et al. 2009). Our group has demonstrated IK is attenuated in CB glomus cells from CHF rabbits (Li et al. 2004; Li & Schultz, 2006), and that hypoxic inhibition of these channels is enhanced in CHF (Li & Schultz, 2006). The depression in IK from the CB glomus cells in CAO rabbits is consistent with that observed in CHF, and likely to contribute to the enhanced CB afferent discharge activity and chemoreflex function observed in both CAO and CHF rabbits. However, we did not test the responsiveness of IK to hypoxia in the present study.

While these results suggest that altered KV channel activity contributes to enhanced glomus cell excitability in CHF and in response to chronic reductions in CB blood flow, other ion channels also are likely to be involved. For example, we recently described enhanced voltage-gated Ca2+ currents in CB glomus cells in CHF (Li et al. 2010). Clearly, many ion channels and channel modulators are known to influence glomus cell excitability (Prabhakar, 2000; Kumar, 2007; Fitzgerald et al. 2009; Nurse, 2010) that have yet to be studied in CHF or CAO conditions.

Role of NOS–NO pathway in CB chemoreflex hypersensitivity in CAO and CHF

Our previous studies have found that a decrease in NO production in the CB is involved in the enhanced CB chemoreceptor activity and peripheral chemoreflex function in CHF rabbits (Sun et al. 1999a; Li et al. 2004, 2005). Inhibition of NOS activity enhances RSNA in response to hypoxia in sham but not in CHF rabbits and administration of NO donor blunts the RSNA response in CHF but not in sham rabbits (Ding et al. 2008). Furthermore, a specific nNOS inhibitor enhanced CB chemoreceptor activity in sham rabbits and failed to increase CB chemoreceptor activity in CHF rabbits (Li et al. 2005). Immunofluorescence labelling for nNOS, nNOS protein and mRNA expressions are markedly lower in CB tissues from CHF rabbits compared with that in sham rabbits (Li et al. 2005, 2010; Ding et al. 2008).

As in CHF, CAO in the present study decreased nNOS expression and NO levels in the CB resulting in increased CB chemoreceptor activity and chemoreflex function, since these effects could be reversed by administration of an NO donor. These results, taken together, demonstrate that a marked downregulation of endogenous nNOS and NO levels in the CB is induced by chronic reduction of CB blood flow and likely contributes to enhanced CB chemoreceptor activity in CHF.

Role of Ang II system in CB chemoreceptor hypersensitivity in CAO and CHF

In addition to the NO signalling pathway, the renin–angiotensin system is markedly activated and Ang II is elevated in patients and animals with CHF (Francis, 1989; Roig et al. 2000; van de Wal et al. 2006). Furthermore, Ang II has been confirmed to increase CB chemoreceptor activity via AT1-R (Allen, 1998). A local Ang II system is functional in rat CB (Lam & Leung, 2002) and rabbit (Li et al. 2006). We have demonstrated that CHF increases Ang II production and AT1-R protein expression in CB, which contribute to CHF-enhanced chemoreceptor activity (Li et al. 2006, 2007). Similarly, endogenous Ang II concentration and AT1-R expression are increased in the CB of CAO rabbits. Importantly, this CB tissue Ang II–AT1-R mechanism contributes to the enhanced CB chemoreceptor sensitivity and chemoreflex function in this model as demonstrated by reversal of these effects by the AT1R antagonist losartan. Based on these results, locally produced Ang II is another important factor in the modulation of CB chemoreceptor function through the AT1-R in the CAO state, and reduced CB blood flow is likely to be the trigger to increase Ang II system in CHF CB.

Perspectives

A chronic reduction of tissue blood flow is a hallmark of CHF. The present investigation provides compelling evidence that the changes in CB function seen in CHF are likely to be caused or markedly influenced by chronic reductions in CB blood flow. CB chemoreflex activation of sympathetic activity is one of several interacting afferent and central neuronal pathways causing autonomic imbalance in CHF. This study raises the question whether impaired blood flow may contribute also to alterations seen in other autonomic pathways in CHF.

These results have important clinical implications. Whereas improved blood flow is an important end point in the treatment of CHF, improved blood flow itself may be effective in limiting the progression of the disease. Indeed the beneficial effects of exercise on autonomic function in CHF (Li et al. 2008; Gao et al. 2007) may be rooted in the effect of exercise to increase blood flow.

The functional link between CB blood flow and CB chemoreceptor activity remains an enigma that needs to be further addressed. Our results and those of others (Biscoe et al. 1970; Lahiri, 1980) suggest that this link is not likely to be oxygen delivery to the CB, since acute reductions of CABF have no effect on CB chemoreceptor function. We speculate, rather, that flow-mediated changes in CB endothelial cell function may be involved. This concept is consistent with potential flow-mediated endothelial effects on the Ang II and NO signalling mechanisms within the CB. Further studies will be required to address this hypothesis.

In summary, the present study demonstrates that CHF reduces blood flow to the CB, and the same degree of flow reduction induced by CAO mimics an enhanced peripheral chemoreflex function, CB chemoreceptor hyperactivity and depression of IK in CB glomus cells as observed in CHF. CAO also upregulates the ANG II–AT1-R system and downregulates nNOS–NO signalling pathway in the CB, which are similar to alterations in the CB induced by CHF. Our results suggest that a chronic reduction in CB blood flow contributes to CHF-enhanced CB chemoreflex function.

References

  1. Top of page
  2. Non-technical summaryAbstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Appendix

  1. Top of page
  2. Non-technical summaryAbstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Author contributions

This study was performed in the Department of Cellular and Integrative Physiology at the University of Nebraska Medical Center, Omaha, NE, USA. All authors contributed to the conception and design of the experiments, collection, analysis and interpretation of data, and drafting the article. All authors approved the manuscript as submitted. There is no conflict of interest.

Acknowledgements

This study was supported by a Program Project Grant from the Heart, Lung and Blood Institute of NIH (PO1-HL62222) and a fellowship for Y. Ding from the American Heart Association, Heartland Affiliate (0725749Z). The authors wish to thank Kaye Talbitzer for her surgical assistance and management of the heart failure animal core at University of Nebraska Medical Center, Mary Ann Zink and Xinying Niu for their technical support.